1. Field of the Invention
The invention is in the field of mammalian reproduction and reproductive immunology and is directed to methods for detecting or isolating acrosome-reacted sperm and mature oocytes which are useful in diagnosis of infertility, for enhancement of in vitro fertilization, and for fertilization inhibition in vivo.
2. Description of the Background Art
Mammalian reproduction is a highly complex biological process which involves diverse specialized molecular systems. The importance of an immunological approach to reproduction stems from basic and clinical studies which demonstrated that reproductive cells and tissues express unique or shared molecules that can elicit immune responses, and that such immune responses can contribute to infertility.
Antibodies to Antigens of the Reproductive Tissues
One important approach to delineating the role of the immune system in reproduction has been the production and characterization of monoclonal antibodies (mAbs) for the identification, isolation, and characterization of mammalian, especially human, reproductive tissue antigens, and the use of such antibodies to block biological activities important in the reproductive process. (See, for example, Anderson, D. J. et al., J. Reprod. Immunol. 10:231-257 (1987)).
A number of mouse mAbs, such as, for example, H316, raised against syncytiotrophoblast plasma membranes from term human placentae, recognize the conserved region of a polymorphic cell surface molecule expressed by all human trophoblast populations, as well as peripheral blood leukocytes, certain specialized epithelia-including endometrial glands, and a number of malignant cell types (Johnson, P. M. et al., Amer. J. Reprod. Immunol. 1:246-254 (1981); McLaughlin et al., Int. J. Cancer 30:21 (1982); Bulmer et al., Placenta 6:127-40 (1985); Stern et al., J. Immunol. 137:1604 (1986)). The tissue distribution and biochemical characteristics of the H316-reactive antigen suggested a close relationship to the "TLX" (trophoblast/leukocyte-common or cross-reactive) antigen family, which had been speculated to induce blocking factors and recruit suppressor cells to the fetal-maternal interface during normal pregnancy. Polyclonal anti-TLX xenoantisera, which originally were used to characterize this antigen system, also react with human semen (Faulk et al., Immunol. Rev. 75:139 (1983); Bulmer et al., supra; Stern et al., supra)).
It has been speculated that the TLX antigen system, which may be recognized by the H316 antibody (Bulmer et al., supra; Stern et al., supra) plays a central role in the regulation of maternal-fetal immunological interactions (Faulk et al., supra). However, these regulatory mechanisms have not been clearly defined in pregnancy, nor have they been investigated in other cellular systems expressing these antigens.
The H316 mAb was included in a panel of anti-sperm monoclonal antibodies recently evaluated in a workshop sponsored by the World health Organization (results reported in: Anderson et al. (1987), supra). This mAb (Workshop #S60) and another mAb included in a panel of anti-trophoblast mAbs, GB24 (Workshop #T22), from Dr. B. L. Hsi, Nice, France, appeared to react with the same antigen. H316 was found in this workshop to react with the acrosomal region of methanol-fixed epididymal and ejaculated sperm, as well as with sperm and germ cells in methanol-fixed frozen sections of adult and fetal human testis. The antibody did not react by radioimmunoassay (RIA) or immunofluorescence (IF) assays with surface antigens of unfixed, freshly ejaculated motile sperm. However, the antibody did react with acrosomal surface antigens on a majority of live motile sperm following either an 8 hour incubation with F-10 capacitation medium, or a 30 minute incubation in medium containing the calcium ionophore, A23187, which also induces sperm acrosome reactions.
Complement Binding Proteins
A family of regulatory proteins that bind to and regulate various of the complement (C) proteins and peptides has been described (Holers, V. et al. Immunol. Today 6:188-192(1985); Reid, et al., Immunol. Today 7:230-223 (1986); Lublin, D. M. & Atkinson, J. P., Ann. Rev. Immunol. 7:35-58 (1989)). Among the C regulatory proteins are the receptor for C3b known as CR1, factor H of the alternate pathway, the C3b inactivator (I), decay accelerating factor (DAF), and gp45-70. The abbreviation CR will be used herein to signify C receptors and C regulatory proteins.
CR1 is an .sup..about. 200 kD intrinsic membrane glycoprotein found on human erythrocytes, granulocytes, B lymphocytes, some T lymphocytes, monocytes, macrophages and glomerular podocytes. The role of CR1 in C-mediated endocytosis (Fearon, D. T. et al., J. Exp. Med. 153:11615-1628 (1981)) and phagocytosis (Griffin, F. M. Jr. et al., J. Exp. Med. 154:291-305 (1981); Wright, S. D. et al., J. Exp. Med. 156:1149-1164 (1982)) is well known.
The local activation and deposition of C components is regulated in part by acceleration of the decay of the multi-component complex known as C3 convertase, which is a necessary intermediary in C activation by the classical pathway and the alternate pathway. DAF, recognized for its role in accelerating decay of C3 convertase, is a 70 kD single chain intrinsic membrane glycoprotein present on erythrocytes, platelets and leukocytes. DAF binds with high affinity to C3b/C4b molecules only when they are in the membrane of the same cell as DAF, and its primary role is thought to be the prevention of the assembly of the convertase complex, rather than the dissociation of already formed complexes (Medof, M. E. et al., Complement 1:168 (1984)). CR1 also shares this decay accelerating activity.
Gp45-70 is identical to the membrane cofactor protein (MCP), which was named for its action as a co-factor in the factor I-dependent cleavage of C3b (Seya, T. et al., J. Exp. Med. 163:837-855 (1986)). This glycoprotein normally has a characteristic doublet protein band upon polyacrylamide gel electrophoresis (PAGE), with mean molecular weights of 58 and 63 kD. MCP was characterized as a cell-associated protein on the human monocyte-like cell line, U937, has cofactor activity but is not a classical C3b receptor. Antisera to human CR1, DAF, H, and two other C receptors, CR2 and CR3, do not cross react with MCP. The expression of CR1 and MCP on the cell surface appears to be reciprocal. MCP has been detected on human T and B lymphocytes, monocytes, platelets, epithelial cells, fibroblasts, and mononuclear-derived cell lines. The activity profiles of MCP and DAF are largely non-overlapping and complementary, leading to the suggestion that they act synergistically to prevent C activation on autologous tissues (Seya et al., supra).
More recently, the cDNA for MCP has been found to encode a protein having 4 short concensus repeats of about 60 amino acids rich in cysteine, the pattern of which is found in all C regulatory proteins studied. THe gene for MCP has been mapped to human chromosome 1 (q32), within 150 kilobases of the 3' end of the CR1 gene. The MCP cDNA also encodes two serine/threonine-reich regions, known sites for 0-glycosylation (Lublin, D. M. et al., J. Exp. Med. 168:181-194 (1988); Post, T. W. et al., FASEB J. 3:A368 (1989) (abstr. #828)).
The link between MCP and fertility is discussed below.
Infertility
A large number of cases of human infertility, especially in the male, are not currently amenable to accurate diagnosis and the responsible mechanisms remain undetected. Indeed there is no practical gold standard for diagnosing male infertility. Clearly, multiple alterations in sperm anatomy and physiology can produce infertility. An urgent need exists in the art for new tests for diagnosing male and female infertility of various unknown origin. The invention disclosed herein is directed to such a need.
In Vitro Fertilization and Gamete Intrafallopian Tube Transfer
In vitro fertilization (IVF) and embryo transfer (ET), and gamete intrafallopian tube transfer (GIFT) wherein actual fertilization occurs in vivo, are gaining popularity as therapies for human infertility. The success of these procedures relies on various factors, not the least of which is the ability of the in vitro conditions, including the constitution of the synthetic medium, to promote the fertilization process. The media employed in these procedures were borrowed from other fields of tissue culture. It is thought that as the synthetic media more closely mimic the natural environment, the efficacy of IVF increases (Quinn, P. J. et al., Fertil. Steril. 44:493 (1985); Quinn, P. J., International Patent Publication WO 86/07377 (1986); Demir, R. H., Mt. Sinai J. Med. 56:141-146 (1989)). Considering the complexity of ovarian follicular fluid, fallopian tubal fluid, uterine and cervical mucus, and seminal fluid, there is an urgent need in the art to understand the constituents of these fluids and their respective roles in fertilization.
Complement and Fertilization
Oliphant and his colleagues (see, for example, Cabot, C. L. & Oliphant, G., Biol. Reprod. 19:666-672 (1978) and Suarez, S. S. & Oliphant, G., Biol. Reprod. 27:473-483 (1982)) have disclosed the presence of C components in bovine follicular fluid and their potential role in the reproductive process. For example, Cabot et al. (supra) demonstrated that antibodies to bovine C3 inhibited the ability of follicular fluid to induce the acrosome reaction in sperm, and suggested that the alternate pathway of C activation was involved in sperm capacitation. Suarez et al. (supra) found that C1q binds to washed ejaculated rabbit sperm, indicating the presence of bound immunoglobulins (i.e. antibodies). In the presence of serum containing active C components, C1q binding led C activation and ultimate damage and acrosomal loss. This potentially destructive process was shown to be inhibited by factors present in seminal plasma (Eng, L. A. & Oliphant, G., Biol. Reprod. 19:1083-1094 (1978)). It was therefore proposed that seminal plasma factors protect sperm both in the male reproductive tract and in proximal parts of the female reproductive tract (Suarez et al., supra) from C-mediated damage.
Bedford and Witkin (J. Reprod. Fert. 69:523-528 (1983)) reported that depletion of C in female rabbits in vivo before mating by treatment with cobra venom factor did not compromise the occurrence of the acrosome reaction after mating. This observation argues that the acrosome-reaction does not depend on C. However, C depletion resulted in a higher proportion of eggs fertilized, implying that C-mediated events may normally exert an inhibitory effect on sperm in the female reproductive tract.
Fahmi, H. A. et al. (J. Dairy Sci. 683318-3322 (1985)) showed that C levels (measured as total hemolytic C) in bovine follicular fluid varied over a 2-5 fold range with the estrus cycle, being highest at estrus and metestrus. At its peak, follicular C levels exceeded serum C levels by 5-22 fold. C3 production by uterine epithelial cells also appears to be under hormonal regulation. C3 transcript and protein levels increased after estradiol treatment in uterine epithelial cells, and this effect was inhibited by progesterone (Lyttle, C. R. et al., Complement Workshop, 1989)).
Tauber, P. F. et al. (Am J. Obstet. Gynecol. 151:1115-1125 (1985)) examined secretory proteins of the cervical, uterine, and fallopian tube mucosa in humans, and disclosed that relatively low levels of diffusible total C activity, and of C3 proteins, were present in these fluids. The levels varied both within and between organs. No role for C in the reproductive process was postulated, and a potential role for C as an anti-bacterial defense in the female reproductive tract was even discounted.
C has been postulated to play a role in endometriosis, a condition associated with female reproductive failure. Isaacson, K. B. et al. (J. Clin. Endocrinol. Metab. 69:1003-1009 (1989)) found that glandular epithelium of endometriotic tissue synthesized and secreted C3, which could be responsible directly, via its fragments or via other C activation products, for many of the immunological and inflammatory phenomena well-known in endometriosis.
In summary, the function of C, its components, or any of its receptors, binding or regulatory proteins, in the facilitation or inhibition of fertilization remains unknown. An understanding of how, when, and where C or C components function in reproductive processes, and an application of this knowledge to regulation and facilitation of fertilization, is a fundamental problem in the art to which the present invention is addressed.